Background MicroRNAs (miRs) and transfer RNA fragments (tRFs) regulate diverse brain processes, from neurogenesis to neurological disease. While both these small RNA classes have been implicated in cell-type-specific regulation, most studies leaned on bulk measurements, due to technical limitations in detecting small RNAs using single-cell RNA sequencing. Consequently, the cell-type specificity of brain miRs and tRFs remains poorly characterized, limiting functional interpretation.

Results To generate a comprehensive, cell-type-resolved atlas of miRs and tRFs from live human brain tissues, we isolated neurons, astrocytes, microglia, and oligodendrocytes from neurosurgery-derived fresh human brain samples and profiled their small RNAs by RNA-sequencing. This revealed pronounced cell-type-specific differences in miR and tRF levels, identified novel small RNA cell-type markers, and provided insight into the genetic regulation of miR cell-type specificity. Notably, neurons exhibited elevated levels of 5′ tRNA halves compared to glial cells, including enriched fragments derived from glycine, leucine and lysine tRNAs. Finally, our resource enabled assignment of cell-type origins to miRs associated with healthy brain aging and tRFs linked to Alzheimer’s disease.

Conclusions Highlighting the importance of cellular context for data interpretation, both human brain miRs and tRFs exhibit pronounced cell-type-specific differences associated with brain aging and neurodegenerative diseases. Combined with an accompanying statistical tool for miR cell-type enrichment analysis, this work provides a publicly available resource for resolving the cell-type origins of small RNAs in the human brain.